Studies of Plasmodium falciparum dihydrofolate-reductase thymidylate synthase: potential advantages.
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Amino Acid Sequence↗
Biomedical subjects
J Inselburg
Publications and source records attributed to J Inselburg.
Plasmodium falciparum: three amino acid changes in the dihydrofolate reductase of a pyrimethamine-resistant mutant.
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Amino Acid Sequence↗
Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.
We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys.
Amino Acid Sequence↗
Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.
A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts.
Amino Acid Sequence↗
Study of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum.
The production of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional protein of Plasmodium falciparum was measured in the wild type, pyrimethamine sensitive strain, FCR3, and in a pyrimethamine resistant mutant, FCR3-D7, which contains a DHFR-TS gene duplication that overproduces a mutant enzyme. The DHFR-TS content in both strains began to increase significantly from the early trophozoite stage through schizogony. The DHFR-TS content in either the ring or trophozoite-schizont stage parasites remained constant for at least 9 hr in the presence of protein synthesis-inhibitory levels of cycloheximide, which suggested that the measure of enzyme accumulation was a measure of enzyme synthesis. Actinomycin D treated parasites did not accumulate DHFR-TS which suggested that the DHFR-TS mRNA had a short half-life. DHFR-TS accumulated in the presence of aphidicolin inhibition of DNA synthesis which indicated that both syntheses could be uncoupled.
Animals↗
Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication.
The accumulation of [3H]pyrimethamine by pyrimethamine-resistant (Pyrr) mutants of the Plasmodium falciparum strain FCR3 was examined by measuring the accumulation of drug in infected red blood cells. [3H]Pyrimethamine was stage specifically accumulated in trophozoites and schizont infected red blood cells. The mutant parasites accumulated drug as efficiently as FCR3. Pyrimethamine was associated with a high molecular weight protein that eluted from a Sephadex G200 column exactly as [3H]fluorodeoxyuridinemonophosphate (FdUMP) labeled parasite dihydrofolate reductase-thymidylate synthetase (DHFR-TS) enzyme. These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane. DHFR-TS-[3H]FdUMP enzyme complex of all the Pyrr mutants and FCR3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One highly resistant mutant, FCR3-D7, exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of DHFR-TS protein than FCR3 but only a normal level of DHFR activity. The genomic DNA of FCR3-D7 was shown to contain at least twice as much DHFR-TS specific DNA than either FCR3-D8, another Pyrr mutant, or FCR3. Preliminary results suggested some of the DHFR-TS genetic material in FCR3-D7 is associated with a gene duplication.
Animals↗
Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.
Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes.
Amino Acid Sequence↗
Plasmodium falciparum-inhibitory monoclonal antibodies produced by human hybridomas.
Stable human hybridomas were generated that produced inhibitory anti-Plasmodium falciparum monoclonal antibodies. Peripheral blood lymphocytes, obtained from adults in Liberia, a malaria endemic area, were immortalized with Epstein-Barr virus and then fused with KR4, a human, lymphoblastoid cell line. Stable hybridomas that produced anti-P. falciparum monoclonal antibody were identified by an ELISA assay that used the trophozoite and schizont antigens of both the Honduras I and FCR3 parasite strains. Monoclonal antibodies produced by selected hybridomas derived from lymphocytes of two individuals were subsequently studied. The anti-parasite antibodies were produced at 1-3 micrograms/ml in culture supernatants. All of the monoclonal antibodies bound specifically to trophozoites and schizonts of both strains of parasite in an indirect immunofluorescence assay and inhibited production of ring stage parasites by more than 90% when added to trophozoite or schizont containing erythrocytes in culture. Western immunoblot analysis of antigens obtained from trophozoites and schizonts (parasite age span of 36 to 48 h) was performed using either affinity purified or ammonium sulfate-concentrated monoclonal antibody. Antibody from three hybridomas which bound primarily to antigens of the Honduras 1 strain had Mr of approximately 140,000, 130,000 and 123,000.
Animals↗
Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants.
We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.
Drug Resistance, Microbial↗
Isolation of temperature-sensitive mutants of Plasmodium falciparum.
Temperature-sensitive mutants of a malaria parasite, Plasmodium falciparum, have been isolated. A culture of parasites was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and repeatedly subjected to a selective pressure designed to favor the enrichment in the culture of parasites that either grew more slowly or ceased growing at 39 degrees C. Cloned parasites derived from cultures subjected to selective pressure were subsequently examined for their ability to grow at 38.5, 39 and 39.5 degrees C. Two temperature-sensitive mutants were isolated that exhibited reduced growth at 39.5 degrees C and one of them also exhibited reduced growth at 39 degrees C. A previously described aphidicolin-resistant mutant of P. falciparum also exhibited reduced growth at 39 degrees C.
Animals↗
Isolation and characterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies.
Three mouse hybridomas were isolated that produced IgM monoclonal antibodies (Mab) which reacted with erythrocytic stages of Plasmodium falciparum and inhibited the invasion of erythrocytes in vitro. Those Mab, initially identified by an ELISA screening of hybridoma culture medium, exhibited a strong binding to trophozoite and schizont but not to ring or merozoite stage parasites or to erythrocytes in an indirect immunofluorescence assay. All inhibited the parasite's ability to infect erythrocytes in an in vitro invasion inhibition assay. Western blot analysis of the binding of the Mab to SDS-PAGE-separated parasite antigens isolated from the ring, trophozoite, schizont or spontaneously released merozoite stages, indicated that two of the Mab bound to a Mr 105,000 antigen in trophozoites and schizonts while the third Mab did not. All three Mab also bound to Mr 30,000-40,000 antigens in all stages, however, in all instances binding to these antigens was enhanced in merozoites. It was further observed that the two Mabs that bound to a Mr 105,000 antigen: exhibited a markedly reduced binding to the Mr 105,000 antigen in merozoite preparations; exhibited different relative intensities of binding to the trophozoite and schizont antigens; both bound to the same Mr 105,000 antigen as demonstrated through Western blot analysis of antigens separated by two-dimensional gel electrophoresis. The findings suggest that the inhibitory Mab bound to different epitopes of the same antigen and that the antigen may either be processed or degraded at about the time of merozoite release and erythrocyte invasion.
Animals↗
Induction and isolation of artemisinine-resistant mutants of Plasmodium falciparum.
Five artemisinine-resistant mutants of Plasmodium falciparum have been isolated from a cloned parent following mutagenic treatment with N-methyl-N'-nitronitrosoguanidine. This observation may influence the strategy of the use of artemisinine or its derivatives. Mutant isolation should prove useful in the study of drug resistance to other antimalarials.
Antimalarials↗
Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor.
The asexual development cycle of Plasmodium falciparum, a malarial parasite of humans, has been synchronized in culture by treating ring-stage parasites with aphidicolin, an inhibitor of DNA synthesis. Optimization of both the concentration of drug added to ring stage containing red blood cells and the duration of exposure of parasites to drug led to a reversible block of their maturation at the early trophozoite stage. Release of the aphidicolin block led to a synchronous development of parasites that was manifested by about 80% of the new ring stages being produced within a 2- to 3-hr interval.
Animals↗
Synthesis of DNA during the asexual cycle of Plasmodium falciparum in culture.
DNA synthesis in cultures of tightly synchronized, developing, asexual forms of Plasmodium falciparum began during the early trophozoite stage between 29.5 and 31 h after parasite invasion and continued through most of the remainder of schizogony. DNA synthesis was inhibited by both hydroxyurea and aphidicolin. Aphidicolin had a biphasic inhibitory effect on DNA synthesis with a portion of the DNA synthesis being considerably more resistant to inhibition. These agents did not block the morphological maturation of ring forms to early trophozoites but did block further maturation of trophozoites and schizonts.
Age Factors↗
Induction and selection of drug resistant mutants of Plasmodium falciparum.
Conditions for mutagenizing Plasmodium falciparum in culture with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine have been established. Parasite mutants resistant to aphidicolin, cycloheximide and sinefungin have been selected and subcloned.
Adenosine↗
Stage-specific inhibitory effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum.
The effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum grown in vitro was examined over a wide range of concentrations. Cyclic AMP inhibited both processes in a stage-specific manner. Asexual maturation was inhibited from shortly after parasite entry into the red cell through the ring stage. However, trophozoites and schizonts matured normally in the presence of cyclic AMP and produced infectious merozoites. Gametocyte formation was inhibited by 95% when 1.0 mM cyclic AMP was added to synchronously growing parasites in the ring stage of development but was only inhibited by 15% when added in the trophozoite or schizont stages. Cyclic AMP was not found to increase gametocyte formation over a wide range of concentrations.
Animals↗
Gametocyte formation by the progeny of single Plasmodium falciparum schizonts.
Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.
Animals↗
ColE1 copy number mutants.
A deletion mutant of the colicin E1-derived plasmid, pDMS6642, exhibited an approximately fourfold increase in copy number. We subsequently isolated hydroxylamine-induced mutants of that plasmid that had a further increase in copy number. Analysis of them suggests that the increased copy number of pDMS6642 is associated with transcriptional readthrough from a Tn3 transposon into the region of ColE1 containing information that influences plasmid replication. The hydroxylamine mutation in one copy number mutant appeared to increase the plasmid copy number by stimulating readthrough transcription from the Tn3 transposon into the ColE1 replication control region, whereas the other hydroxylamine mutation acts by another mechanism.
Bacteriocin Plasmids↗